Figures and data
Detection of sulfane sulfur in GIF/MT3.
(A) Preparation of recombinant Zn7GIF/MT3 and oxidized apo-GIF/MT3 proteins. Recombinant human Zn7GIF/MT3 (10 μM) was incubated in HCl (0.1 N) at 37°C for 30 min and then replaced with 20 mM Tris-HCl (pH 7.5) buffer and incubated for 36 h at 37°C. After removal of low-molecular-weight molecules using 3 kDa centrifugal filtration, GIF/MT3-bound zinc content was measured using ICP-MS. (B) FT-ICR-MALDI-TOF/MS spectrum (positive-ion mode) of Zn7GIF/MT3. (C) FT-ICR-MALDI-TOF/MS spectrum (positive-ion mode) of oxidized apo-GIF/MT3. (D) Raman spectra of Zn7GIF/MT3, oxidized apo-GIF/MT3, and HPE-IAM-treated GIF/MT3 in the 250–800 cm−1 region. Optimized geometries for (E) α-domain and (F) β-domain models of apo-GIF/MT3 (assuming some cysteines with persulfide and tetrasulfide bonds as shown). Optimized geometries for (G) α-domain and (H) β-domain models of apo-GIF/MT3 (assuming some cysteines with disulfide bonds). (I) Calculated Raman spectra of apo-GIF/MT3 models with/without sulfane sulfurs. FT-ICR, Fourier transform ion cyclotron resonance.
Sulfane sulfur assay optimization and quantification of MT sulfane sulfur content.
(A) Schematic showing the detection of sulfane sulfur in Zn7GIF/MT3. (B) Sulfane sulfur detected in Zn7GIF/MT3 after incubation with the indicated concentrations of HPE-IAM at 60°C for 36 h. (C) Sulfane sulfur detected in Zn7GIF/MT3 after incubation with 5 mM HPE-IAM at 60°C for 36 h. (D) Sulfane sulfur detected in Zn7GIF/MT3 after incubation with 5 mM HPE-IAM at 37°C or 60°C for the indicated times. (E) Sulfane sulfur detected in human Zn7MT1, Zn7MT2, Zn7GIF/MT3, wild-type (WT) Zn7GIF/MT3, and apo-GIF/MT3 with all Cys residues mutated to Ala (all C/A), each incubated with 5 mM HPE-IAM at 60°C for 36 h. Sulfane sulfur content was measured using LC-MS/MS. HPE-IAM, β-(4-hydroxyphenyl)ethyl iodoacetamide.
Sulfane sulfur stability in apo-GIF/MT3 and its restoration by a reducing agent.
(A) Stability of sulfane sulfur in apo-GIF/MT3 incubated with or without (Cont) zinc. To prepare apo-GIF/MT3, Zn7GIF/MT3 was incubated in 0.1 M HCl for 30 min, then the buffer was replaced with 20 mM Tris-HCl (pH 7.5). To examine the stability of sulfane sulfur in apo-GIF/MT3, freshly prepared apo-GIF/MT3 (2 µM) with or without added zinc ions were incubated at 37°C for up to 24 h. (B) Effect of tris(2-carboxyethyl)phosphine (TCEP) on sulfane sulfur binding and free SH/SSH groups in oxidized apo-GIF/MT3. To prepare oxidized apo-GIF/MT, Zn7GIF/MT3 was incubated in HCl (0.1 N) at 37°C for 30 min and then replaced with 20 mM Tris-HCl (pH 7.5) buffer and incubated for 36 h at 37°C. The resulting oxidized apo-GIF/MT3 protein (10 µM) was incubated with 0, 1, 10, 50, or 100 mM TCEP in 20 mM Tris-HCl (pH 7.5) at 37°C for 1 h, then low-molecular-weight molecules were removed by 3 kDa ultrafiltration for 6 times. Sulfane sulfur content was determined using LC-ESI-MS/MS, and the concentrations of free SH/SSH groups were measured using Ellman’s reagent.
Reactivity of HPE-IAM with tetrasulfide derivatives as models of tetrasulfide bridges in apo-GIF/MT3.
(A) Reactivity of HPE-IAM with N-acetylcysteine (NAC) derivatives. Oxidized NAC (oxiNAC), NAC-trisulfide (NAC-S1), and NAC-tetrasulfide (NAC-S2) (each 10 µM) were incubated with HPE-IAM (5 mM) at 60°C for 1 or 36 h with or without TCEP (1 mM). (B) Reactivity of HPE-IAM with diallyl polysulfide derivatives. Diallyl disulfide (DADS), diallyl trisulfide (DATS), or diallyl tetrasulfide (DATetraS) (each 10 µM) were incubated with HPE-IAM (5 mM) at 60°C for 1 or 36 h with or without TCEP (1 mM). (C) Scheme showing possible reactions of tetrasulfide derivatives with HPE-IAM and TCEP. Bis-S-HPE-AM, bis-S-β-(4-hydroxyphenyl)ethyl acetamide.
Redox-dependent release of zinc ions and recycling of sulfane sulfur in GIF/MT3.
(A) Quantitation of zinc ions released from Zn7GIF/MT3 by H2O2 and S-nitroso-N-acetylpenicillamine (SNAP). To examine the release of zinc ions by H2O2 and SNAP, Zn7GIF/MT3 (10 µM) was treated with H2O2 (1 or 5 mM) or SNAP (1 or 5 mM) in 100 mM Tris-HCl (pH 7.5) at 25°C for 30 min. After removing H2O2/SNAP using 3 kDa ultrafiltration for 4 times, free SH/SSH groups and sulfane sulfur content in GIF/MT3 were determined. (B) Free SH/SSH content in Zn7GIF/MT3, determined by H2O2 or SNAP treatment after incubation with TCEP. To examine the interaction of Zn7GIF/MT3 with H2O2 or NO, Zn7GIF/MT3 (10 µM) was incubated with H2O2 (1 or 5 mM) or SNAP (1 or 5 mM) in 100 mM Tris-HCl (pH 7.5) at 25°C for 30 min. After removing H2O2/SNAP using 3 kDa ultrafiltration for 4 times, the resulting proteins (5 µM) were incubated with TCEP (50 mM) in 100 mM Tris-HCl (pH 7.5) at 37°C for 1 h. After removing TCEP using 3 kDa ultrafiltration for 5 times, sulfane sulfur content was determined using LC-ESI-MS/MS and the concentrations of free SH/SSH groups were measured using Ellman’s reagent. (C) Proposed reactions between a zinc/persulfide cluster in GIF/MT3 and H2O2 or NO.
Contribution of sulfane sulfur in GIF/MT3 to zinc binding.
(A) To eliminate sulfane sulfur in Zn7GIF/MT3 by cyanolysis, Zn7GIF/MT3 (10 µM) was reacted with KCN (75 mM) in 100 mM Tris-HCl (pH 7.5) at 37°C for 14 h. After removal of KCN, the resulting protein was incubated with TCEP (10 mM) in 100 mM Tris-HCl (pH 7.5) at 37°C for 1 h. After removal of TCEP, sulfane sulfur content in GIF/MT3 was determined using LC-ESI-MS/MS. (B) Comparison of zinc binding capacity of GIF/MT3 before and after cyanolysis. Zn7GIF/MT3 (10 µM) was incubated with KCN (75 mM) in 100 mM Tris-HCl (pH 7.5) at 37°C for 14 h. After removal of KCN, the resulting protein was incubated with TCEP (10 mM) in 100 mM Tris-HCl (pH 7.5) at 37°C for 1 h. After removal of TCEP, the resulting protein (5 µM) was incubated with zinc chloride (50 µM) in 50 mM Tris-HCl (pH 7.5) at 37°C for 1 h. Low-molecular-weight molecules were removed using 3 kDa ultrafiltration after each step. Protein-bound zinc content was determined using ICP-MS. *p < 0.05 and **p < 0.01.
Reduction of apo-GIF/MT3 by thioredoxin (Trx) and subsequent regeneration of sulfane sulfur.
(A) Velocity (V) of Trx-catalyzed reduction of oxidized apo-GIF/MT3 substrate (S). Oxidation of NADPH was followed by measuring the absorbance of NADPH at 340 nm. (B) Comparison of substrate reduction by NADPH and the thioredoxin system. (C) NADP+ formation upon incubation of: oxidized apo-GIF/MT3 with Trx/TR, TRP14/TR, or TRP32/TR; and Zn7GIF/MT3 with Trx/TR. (D) Regeneration of sulfane sulfur in oxidized apo-GIF/MT3 after incubation with the Trx/TR system. TR, Trx reductase; TRP14, Trx-related protein 14; TRP32, Trx-related protein 32.
Structural modeling of sulfane sulfur in GIF/MT3 using MOE, and a reaction scheme for sulfane sulfur-based zinc/persulfide cluster.
(A) Comparison of three-dimensional structures of Zn7GIF/MT3 (pink) and Zn7S20GIF/MT3 (green). (B) Cyclohexane-like Zn3Cys9 cluster in the GIF/MT3 homology model, and bicyclononane-like Zn4Cys11 cluster derived from PDB structure 2F5H with (lower) or without (upper) sulfane sulfur. Yellow, orange, and gray spheres indicate cysteine residues, sulfane sulfur, and zinc ions, respectively. (C) Thermostability and zinc binding affinity scores of GIF/MT3 with different numbers of sulfane sulfurs at each cysteine residue. (D) A proposed model for redox-dependent hold-and-release regulation of zinc ions by GIF/MT3.
Thermostability and metal binding affinity scores of growth inhibitory factor (GIF)/metallothionein-3 (MT3) with or without sulfane sulfur. Values were calculated using the Protein Design module of the Molecular Operating Environment (MOE) software.
Separation of sulfane sulfur-binding proteins from mouse brain cytosol using column chromatography.
(A) Diethylaminoethyl Sepharose CL-6B column. (B) Sephacryl S-100 column. (C) Blue Sepharose column. Triangles, closed circles, and dotted lines indicate sulfane sulfur, protein, and NaCl concentrations, respectively. Portions of each fraction were incubated with 5 mM of HPE-IAM in 20 mM Tris (pH 7.5) at 37°C for 30 min and the sulfane sulfur content was determined from the bis-S-HPE-AM adduct concentration measured using LC-MS/MS. Protein concentration was determined using the bicinchoninic acid assay. Isolation of sulfane sulfur-binding protein was performed as described in the Experimental procedures.
